Universal ligation-detection-reaction microarray applied for compost microbes

Abstract

Background: Composting is one of the methods utilised in recycling organic communal waste. The composting process is dependent on aerobic microbial activity and proceeds through a succession of different phases each dominated by certain microorganisms. In this study, a ligation-detection-reaction (LDR) based microarray method was adapted for species-level detection of compost microbes characteristic of each stage of the composting process. LDR utilises the specificity of the ligase enzyme to covalently join two adjacently hybridised probes. A zip-oligo is attached to the 3'-end of one probe and fluorescent label to the 5'-end of the other probe. Upon ligation, the probes are combined in the same molecule and can be detected in a specific location on a universal microarray with complementary zip-oligos enabling equivalent hybridisation conditions for all probes. The method was applied to samples from Nordic composting facilities after testing and optimisation with fungal pure cultures and environmental clones.

Results: Probes targeted for fungi were able to detect 0.1 fmol of target ribosomal PCR product in an artificial reaction mixture containing 100 ng competing fungal ribosomal internal transcribed spacer (ITS) area or herring sperm DNA. The detection level was therefore approximately 0.04% of total DNA. Clone libraries were constructed from eight compost samples. The LDR microarray results were in concordance with the clone library sequencing results. In addition a control probe was used to monitor the per-spot hybridisation efficiency on the array.

Conclusion: This study demonstrates that the LDR microarray method is capable of sensitive and accurate species-level detection from a complex microbial community. The method can detect key species from compost samples, making it a basis for a tool for compost process monitoring in industrial facilities.

Figure 1

Principle of LDR. A schematic picture of the ligation detection reaction (LDR) [19] and hybridisation to the microarray by the zip-code sequences [25].

Figure 2

Probe sensitivity. Boxplots showing the signal distribution of probes detecting different concentrations of template. "bg" is the background distribution in the same subarray as a given template. Yellow triangles denote the 2.5 SD detection limit above the background median. The false positives above the detection limit are from cZip number 17 which was not used in any of the probes.

Figure 3

Effect of ligation cycle number on the probe signals. The distributions of ligation probe signals after A) 40 cycles B) 80 cycles and C) 120 cycles of ligation.

Figure 4

Probe specificity. a. The intensity values of all the probes against individual templates. On the Y-axis, the set of probes present in each reaction. On the X-axis, the template present in a given reaction. The colour coded values are intensity log ratios of LDR/B3. Highest values are expected to be in the diagonal. Three probes, PenCom-A50, KlyMa2-A84 and PichFerm-A67, do not detect any template. Probe AspFum-A58 is slightly non-specific to I. orientalis template. Pezizomycota-A29 is group-specific. b. Scanned images of an example slide with hybridisations of the complementary control probe and two probes specific for Thermomyces lanuginosus phylotype (TherLa in Fig. 4a) The control probe can be seen to hybridise to all of the spots and the specific probe for the corresponding zip-code sequences.